The invention relates to inhibitors of trypsin-like serine proteases of the general formula
which, besides plasmin, also inhibit plasma kallikrein, and to the preparation and use thereof as medicaments, preferably for the treatment of blood loss, especially in hyperfibrinolytic conditions, in organ transplants or cardiac surgical procedures especially with cardiopulmonary bypass, or as constituent of a fibrin adhesive.
Inhibitors of plasmin and plasma kallikrein (PK) have been disclosed. Plasmin is a trypsin-like serine protease and cleaves numerous substrates C-terminally of the basic amino acids arginine or lysine. Plasmin is formed from the zymogen plasminogen by the catalytic action of the plasminogen activators urokinase or tPA. Plasmin substrates include various proteins of the extracellular matrix and basal membrane, for example fibronectin, laminin, type IV collagen or fibrin, but also numerous zymogens such as proforms of the matrix metalloproteases or of the plasminogen activator urokinase. In blood, plasmin is responsible in particular for fibrinolysis by cleaving fibrin into soluble products.
The endogenous plasmin inhibitors include α2-macroglobulin and the serpin α2-antiplasmin. Under certain pathological conditions there may be spontaneous activation of fibrinolysis. In the event of such a hyperplasminemia, not only is the wound-closing fibrin degraded, but there is also formation of anticoagulant fibrinogen degradation products. Serious impairments of hemostasis may arise thereby. Antifibrinolytics used clinically are synthetic amino carboxylic acids such as ε-aminocaproic acid, p-aminomethylbenzoic acid or tranexamic acid (trans-4-(aminomethyl)cyclo-hexanecarboxylic acid). These compounds block the binding of the zymogen plasminogen to fibrin and thus inhibit activation thereof to plasmin. These compounds are therefore not direct inhibitors of plasmin and are unable to inhibit plasmin which has already been formed. A further antifibrinolytic employed is aprotinin (Trasylol®, Bayer AG, Leverkusen), a polypeptide of 58 amino acids which is obtained from bovine lung. Aprotinin inhibits plasmin with an inhibition constant of 1 nM, but is relatively nonspecific and also effectively inhibits trypsin (Ki=0.1 nM) and plasma kallikrein (Ki=30 nM). Aprotinin also inhibits other enzymes, although with reduced activity.
A main use of aprotinin serves to reduce blood loss, especially in cardiac surgical procedures with cardiopulmonary bypass (CPB), thus distinctly reducing the need for perioperative blood transfusions (Sodha et al., 2006). In addition, aprotinin is also employed in other operations, for example in organ transplants, to inhibit blood loss, or is used as addition in fibrin adhesives.
The use of aprotinin has several disadvantages. Since it is isolated from bovine organs, there is in principle the risk of pathogenic contamination and allergic reactions. The risk of an anaphylactic shock is relatively low with the first administration of aprotinin (<0.1%), but increases on repeated administration within 200 days to 4-5%.
It was recently reported that administration of aprotinin in direct comparison with ε-aminocaproic acid or tranexamic acid induces an increased number of side effects (Mangano et al., 2006). Administration of aprotinin led to a doubling of the number of cases of kidney damage, making dialysis necessary. Likewise, the risk of myocardial infarction and apoplectic stroke was increased through administration of aprotinin by comparison with the control groups.
To date only a few synthetic inhibitors of plasmin have been disclosed. Sanders and Seto (1999) described 4-heterocyclohexanone derivatives with relatively weak activity, with inhibition constants of ≧50 μM for plasmin. Xue and Seto (2005) reported on peptidic cyclohexanone derivatives with IC50 values of ≧2 μM, but further development thereof is unknown. Okada and Tsuda described various derivatives with a 4-aminomethylcyclohexanoyl residue which inhibit plasmin with IC50 values of ≧0.1 μM, but clinical use of these inhibitors is not known (Okada et al., 2000; Tsuda et al., 2001).
Inhibition constants for plasmin have been published in numerous publications on the development of inhibitors of coagulation proteases as antithrombotics, where the aim in these cases was to inhibit plasmin as weakly as possible. A possible use of these compounds for reducing blood loss in cardiac surgical procedures was not mentioned in any of these papers. Thus, for example, the thrombin inhibitor melagatran inhibits plasmin with a Ki value of 0.7 μM, whereas the structurally closely related compounds H317/86 has an inhibition constant of 0.22 μM for plasmin (Gustafsson et al., 1998). However, both compounds inhibit the protease thrombin distinctly more strongly with Ki values of ≦2 nM, and thus administration of melagatran results in strong anticoagulation.
As described in the introduction, aprotinin inhibits not only plasmin but also plasma kallikrein (PK). PK is a multifunctional, trypsin-like serine protease for which several physiological substrates are known. Thus, PK is able to release by proteolytic cleavage the vasoactive peptide bradykinin from high molecular weight kininogen and to activate the zymogens coagulation factor XII, pro-urokinase, plasminogen and pro-MMP 3. It is therefore assumed that the PK/kinin system has an important role in various symptoms, for example in thromboembolic situations, disseminated intravascular coagulation, septic shock, allergies, the postgastrectomy syndrome, arthritis and ARDS (adult respiratory distress syndrome) (Tada et al., 2001).
Accordingly, aprotinin inhibits, by its inhibitory effect on PK, the release of the peptide hormone bradykinin. Bradykinin has, via activation of the bradykinin B2 receptor, various effects. The bradykinin-induced release of tPA, NO and prostacyclin from endothelial cells (see review paper by Schmaier, 2002) influences fibrinolysis, blood pressure and the inflammatory event. It is suggested that systemic inflammatory processes which may occur as side effect in operations are reduced by inhibiting bradykinin release.
Various bisbenzamidines such as pentamidine and related compounds, and esters of ω-amino- and ω-guanidinoalkylcarboxylic acids with micromolar Ki values have been described as PK inhibitors (Asghar et al., 1976; Muramatu and Fuji, 1971; Muramatu and Fuji, 1972; Ohno et al., 1980; Muramatu et al., 1982; Satoh et al., 1985; Teno et al., 1991).
The first selective competitive inhibitors, which are derived from arginine or phenylalanine, were developed by Okamoto et al., (1988) and inhibit PK with Ki values around 1 μM. Several papers on the development of competitive PK inhibitors have been published by the Okada group, with the most active compounds, which are derived from trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanilide, having inhibition constants around 0.5 μM (Okada et al., 1999; Okada et al., 2000, Tsuda et al., 2001). It is common to the said PK inhibitors that they have a relatively high Ki value. U.S. Pat. No. 6,472,393 described potent PK inhibitors with inhibition constants around 1 nM and having a 4-amidinoaniline as P1 residue. PK inhibitors have also been described in U.S. Pat. No. 5,602,253. US 2006/0148901 described PK inhibitors whose inhibitory effect on plasmin is, however, relatively small, these inhibitors differing thereby from the inhibitors described in the present application.
The invention is therefore based on the object of providing low molecular weight active substances which are suitable for therapeutic applications and which reversibly and competitively inhibit in particular plasmin and plasma kallikrein with high activity and specificity and are therefore suitable for hemostasis in various applications, for example in cardiac surgical procedures with CPB, in organ transplants or other operations. A further advantage of these compounds is that through their effect as inhibitor of plasma kallikrein in addition kinin release is reduced and thus kinin-mediated inflammatory reactions can be suppressed. The kinin-induced release of tPA from endothelial cells is in turn suppressed by the inhibited kinin release, it being possible thereby for fibrinolysis to be downregulated by this mechanism. A further advantage of these compounds is, despite selectivity, a certain inhibitory effect of these compounds on FXa and/or thrombin, and thus thrombotic complications are additionally to be reduced on use of these compounds.